scholarly journals Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 423-432 ◽  
Author(s):  
E Cramer ◽  
KB Pryzwansky ◽  
JL Villeval ◽  
U Testa ◽  
J Breton-Gorius
Keyword(s):  
Human Peripheral Blood ◽  
Ultrastructural Localization ◽  
Human Neutrophils ◽  
Cell Fractionation ◽  
Thin Sections ◽  
The Matrix ◽  
Blood Neutrophils ◽  
Microscopic Studies ◽  
Specific Localization ◽  
Monospecific Antibodies

Abstract Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.

scholarly journals Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 423-432 ◽  
Author(s):  
E Cramer ◽  
KB Pryzwansky ◽  
JL Villeval ◽  
U Testa ◽  
J Breton-Gorius
Keyword(s):  
Human Peripheral Blood ◽  
Ultrastructural Localization ◽  
Human Neutrophils ◽  
Cell Fractionation ◽  
Thin Sections ◽  
The Matrix ◽  
Blood Neutrophils ◽  
Microscopic Studies ◽  
Specific Localization ◽  
Monospecific Antibodies

Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.

scholarly journals Electrospun Polydioxanone Loaded With Chloroquine Modulates Template-Induced NET Release and Inflammatory Responses From Human Neutrophils

2021 ◽  
Vol 9 ◽  
Author(s):  
Allison E. Fetz ◽  
Shannon E. Wallace ◽  
Gary L. Bowlin
Keyword(s):  
Growth Factor ◽  
Inflammatory Responses ◽  
Human Peripheral Blood ◽  
Early Time ◽  
Human Neutrophils ◽  
Late Time ◽  
Dependent Manner ◽  
Blood Neutrophils ◽  
Net Release

The implantation of a biomaterial quickly initiates a tissue repair program initially characterized by a neutrophil influx. During the acute inflammatory response, neutrophils release neutrophil extracellular traps (NETs) and secrete soluble signals to modulate the tissue environment. In this work, we evaluated chloroquine diphosphate, an antimalarial with immunomodulatory and antithrombotic effects, as an electrospun biomaterial additive to regulate neutrophil-mediated inflammation. Electrospinning of polydioxanone was optimized for rapid chloroquine elution within 1 h, and acute neutrophil-biomaterial interactions were evaluated in vitro with fresh human peripheral blood neutrophils at 3 and 6 h before quantifying the release of NETs and secretion of inflammatory and regenerative factors. Our results indicate that chloroquine suppresses NET release in a biomaterial surface area–dependent manner at the early time point, whereas it modulates signal secretion at both early and late time points. More specifically, chloroquine elution down-regulates interleukin 8 (IL-8) and matrix metalloproteinase nine secretion while up-regulating hepatocyte growth factor, vascular endothelial growth factor A, and IL-22 secretion, suggesting a potential shift toward a resolving neutrophil phenotype. Our novel repurposing of chloroquine as a biomaterial additive may therefore have synergistic, immunomodulatory effects that are advantageous for biomaterial-guided in situ tissue regeneration applications.

On the ultrastructural localization of cell surface sialyl residues versus anionic sites

1978 ◽  
Vol 36 (2) ◽  
pp. 294-295
Author(s):  
E. Skutelsky ◽  
S. Hoglund ◽  
B. Morein ◽  
E.A. Bayer
Keyword(s):  
Cell Surface ◽  
Ferric Hydroxide ◽  
Ultrastructural Localization ◽  
Anionic Sites ◽  
Successive Treatment ◽  
Coulombic Interaction ◽  
Thin Sections ◽  
Anionic Charge ◽  
Specific Localization

Membrane-associated sialic acids(SA) are considered to have an important role in a variety of cell sur ace interactions. Visualization of SA sites and their ultrastructural quantitative evaluation have heverally been based on the coulombic interaction between anionic sites of sialyl carboxyl groups with polycationic,electron dense markers, e.g. colloidal ferric hydroxide or cationized ferritin(CF). We have recently demonstrated an alternative method, whereby periodate-induced biotinylation (PIB) of unfixed cells can be used for specific localization of SA in thin sections by ferritin-conjugated avidin (FAv). It was further shown that PIB does not affect the surface anionic charge, since the latter is still available to CF-staining. In order to determine the role of anionic sites on the distribution and configuration of cell surface sialoglycoproteins and or sialoglycolipids, we have compared the topographic distribution of attached FAv or CF particles on normal and pathological blood cells,following successive treatment with sodium periodate and biotin hydrazide.

scholarly journals Leukotriene B4 Augments Neutrophil Phagocytosis of Klebsiella pneumoniae

2001 ◽  
Vol 69 (4) ◽  
pp. 2011-2016 ◽  
Author(s):  
Peter Mancuso ◽  
Patrick Nana-Sinkam ◽  
Marc Peters-Golden
Keyword(s):  
Klebsiella Pneumoniae ◽  
Human Peripheral Blood ◽  
Critical Role ◽  
Leukotriene B4 ◽  
Eicosatetraenoic Acid ◽  
Human Neutrophils ◽  
Complement Receptor ◽  
Macrophage Phagocytosis ◽  
Blood Neutrophils ◽  
Cysteinyl Leukotriene Receptor

ABSTRACT Neutrophils play a critical role in the clearance of bacteria from the lung and other organs by their capacity for phagocytosis and killing. Previously, we identified an important role for the leukotrienes in rat alveolar macrophage phagocytosis ofKlebsiella pneumoniae. In this report, we explored the possibility that the leukotrienes play an important role in phagocytosis by neutrophils as well. Inhibition of endogenous leukotriene synthesis by 5-lipoxygenase knockout in mice or by pharmacologic means in human peripheral blood neutrophils attenuated phagocytosis of opsonized K. pneumoniae. Reduced phagocytosis was also observed in human neutrophils pretreated with a leukotriene B4 receptor but not a cysteinyl-leukotriene receptor antagonist. While leukotriene B4 reconstituted defective phagocytosis in leukotriene-deficient neutrophils and enhanced phagocytosis in neutrophils capable of leukotriene synthesis, leukotriene C4, leukotriene D4, 5-hydroperoxyeicosatetraenoic acid, and 5-oxo-eicosatetraenoic acid were ineffective. To determine the opsonin dependence of the leukotriene B4 augmentation of phagocytosis, we assessed the ability of leukotriene B4 to modulate neutrophil phagocytosis and the adherence of sheep erythrocytes opsonized with immunoglobulin G or the complement fragment C3bi. While leukotriene B4 augmented both Fc receptor- and complement receptor-mediated phagocytosis, increased adherence to leukotriene B4-treated neutrophils was limited to complement opsonized targets. In conclusion, we have identified a novel role for leukotriene B4 in the augmentation of neutrophil phagocytosis mediated by either the Fc or complement receptor.

Extracellular ATP stimulates elastase secretion from human neutrophils and increases lung resistance and secretion from normal rat airways after intratracheal instillation

1992 ◽  
Vol 70 (7) ◽  
pp. 1065-1068 ◽  
Author(s):  
M. Flezar ◽  
R. Olivenstein ◽  
A. Cantin ◽  
S. Heisler
Keyword(s):  
Human Peripheral Blood ◽  
Intratracheal Instillation ◽  
Bronchial Tree ◽  
Extracellular Atp ◽  
Brown Norway ◽  
Human Neutrophils ◽  
Lung Resistance ◽  
Intracellular Ca ◽  
Blood Neutrophils

The effect of ATP on intracellular Ca2+ levels and elastase secretion in isolated normal human peripheral blood neutrophils was investigated as was its in vivo effect on lung resistance and mucous secretion. ATP (10−5 M) increased [Ca2+]i from 61 ± 3 to 165 ± 15 nM in nonactivated neutrophils; elastase secretion was increased by 40% from nonactivated neutrophils but was unaffected in fMLP (10−5 M) activated cells. Instillation of ATP (10−5 and 10−3 M) into the airways of brown Norway rats increased both lung resistance and secretion. These findings suggest that aerosolization of ATP into the cystic fibrosis-affected bronchial tree might be hazardous in terms of enhancement of parenchymal damage, which would result from neutrophil elastase release, and in terms of impaired respiratory lung function.Key words: extracellular ATP, respiratory airway, elastase secretion, resistance, pulmonary secretion.

scholarly journals Regulation of intracellular pH in human neutrophils.

1985 ◽  
Vol 85 (3) ◽  
pp. 443-470 ◽  
Author(s):  
L Simchowitz ◽  
A Roos
Keyword(s):  
Intracellular Ph ◽  
Equilibrium Distribution ◽  
Human Peripheral Blood ◽  
Human Neutrophils ◽  
Intracellular Acidification ◽  
Control Value ◽  
Buffering Power ◽  
Blood Neutrophils ◽  
Acid Loading ◽  
Intracellular Alkalinization

The intracellular pH (pHi) of isolated human peripheral blood neutrophils was measured from the fluorescence of 6-carboxyfluorescein (6-CF) and from the equilibrium distribution of [14C]5,5-dimethyloxazolidine -2,4-dione (DMO). At an extracellular pH (pHo) of 7.40 in nominally CO2-free medium, the steady state pHi using either indicator was approximately 7.25. When pHo was suddenly raised from 7.40 to 8.40 in the nominal absence of CO2, pHi slowly rose by approximately 0.35 during the subsequent hour. A change of similar magnitude in the opposite direction occurred when pHo was reduced to 6.40. Both changes were reversible. Intrinsic intracellular buffering power, determined by using graded pulses of CO2 or NH4Cl, was approximately 50 mM/pH over the pHi range of 6.8-7.9. The course of pHi obtained from the distribution of DMO was followed during and after imposition of intracellular acid and alkaline loads. Intracellular acidification was brought about either by exposing cells to 18% CO2 or by prepulsing with 30 mM NH4Cl, while pHo was maintained at 7.40. In both instances, pHi (6.80 and 6.45, respectively) recovered toward the control value at rates of 0.029 and 0.134 pH/min. These rates were reduced by approximately 90% either by 1 mM amiloride or by replacement of extracellular Na with N-methyl-D-glucamine. Recovery was not affected by 1 mM SITS or by 40 mM alpha-cyano-4-hydroxycinnamate (CHC), which inhibits anion exchange in neutrophils. Therefore, recovery from acid loading is probably due to an exchange of internal H for external Na. Intracellular alkalinization was achieved by exposing the cells to 30 mM NH4Cl or by prepulsing with 18% CO2, both at a constant pHo 7.40. In both instances, pHi, which was 7.65 and 7.76, respectively, recovered to the control value. The recovery rates (0.033 and 0.077 pH/min, respectively) were reduced by 80-90% either by 40 mM CHC or by replacement of extracellular Cl with p-aminohippurate (PAH). SITS, amiloride, and ouabain (0.1 mM) were ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Ll-37, the Neutrophil Granule–And Epithelial Cell–Derived Cathelicidin, Utilizes Formyl Peptide Receptor–Like 1 (Fprl1) as a Receptor to Chemoattract Human Peripheral Blood Neutrophils, Monocytes, and T Cells

2000 ◽  
Vol 192 (7) ◽  
pp. 1069-1074 ◽  
Author(s):  
De Yang ◽  
Qian Chen ◽  
Albert P. Schmidt ◽  
G. Mark Anderson ◽  
Ji Ming Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Human Peripheral Blood ◽  
Molecular Size ◽  
Formyl Peptide Receptor ◽  
Human Neutrophils ◽  
Antimicrobial Protein ◽  
Peptide Receptor ◽  
Microbial Invasion ◽  
Formyl Peptide ◽  
Blood Neutrophils

We have previously shown that antimicrobial peptides like defensins have the capacity to mobilize leukocytes in host defense. LL-37 is the cleaved antimicrobial 37-residue, COOH-terminal peptide of hCAP18 (human cationic antimicrobial protein with a molecular size of 18 kD), the only identified member in humans of a family of proteins called cathelicidins. LL-37/hCAP18 is produced by neutrophils and various epithelial cells. Here we report that LL-37 is chemotactic for, and can induce Ca2+ mobilization in, human monocytes and formyl peptide receptor–like 1 (FPRL1)-transfected human embryonic kidney 293 cells. LL-37–induced Ca2+ mobilization in monocytes can also be cross-desensitized by an FPRL1-specific agonist. Furthermore, LL-37 is also chemotactic for human neutrophils and T lymphocytes that are known to express FPRL1. Our results suggest that, in addition to its microbicidal activity, LL-37 may contribute to innate and adaptive immunity by recruiting neutrophils, monocytes, and T cells to sites of microbial invasion by interacting with FPRL1.

scholarly journals Structural and functional heterogeneity among peroxidase-negative granules in human neutrophils: identification of a distinct gelatinase- containing granule subset by combined immunocytochemistry and subcellular fractionation

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3183-3191 ◽  
Author(s):  
L Kjeldsen ◽  
DF Bainton ◽  
H Sengelov ◽  
N Borregaard
Keyword(s):  
Ultrastructural Localization ◽  
Subcellular Fractionation ◽  
Free Form ◽  
Human Neutrophils ◽  
Immunogold Electron Microscopy ◽  
Double Labeling ◽  
Thin Sections ◽  
Dense Granules ◽  
Specific Granules ◽  
Neutrophil Gelatinase

Abstract The existence of separate gelatinase granules in human neutrophils has been a matter of debate in recent years. We have demonstrated that the 135-kD form of neutrophil gelatinase is a complex of 92-kD gelatinase and a novel 25-kD protein termed neutrophil gelatinase-associated lipocalin (NGAL) that, in addition to being complexed with part of the gelatinase, is localized in free form in peroxidase-negative specific granules. Because this association was not appreciated in earlier studies, we decided to reassess the ultrastructural localization of gelatinase using specific antibodies without immunoreactivity towards NGAL. Double-labeling immunogold electron microscopy was performed on frozen thin sections of human neutrophils. Twenty-four percent of all peroxidase-negative granules were labeled with antigelatinase antibody, but not with antilactoferrin antibody. These granules are defined as gelatinase granules. Sixteen percent reacted with antilactoferrin antibody but not with antigelatinase antibody. The rest (60%) reacted with both antibodies. All granules labeling for lactoferrin are defined as specific granules. Gelatinase granules were observed as round and oval forms of considerably smaller size than specific granules, and were less electron dense. Isolated granules obtained by subcellular fractionation were also examined by immunoelectron microscopy. This demonstrated that peroxidase-negative granules comprise a continuum from the most dense granules that contain lactoferrin but no gelatinase to the lightest that contain gelatinase but no lactoferrin. Thus, gelatinase granules do exist as a subpopulation of peroxidase-negative granules and may allow for exocytosis of gelatinase during neutrophil diapedesis without substantial mobilization of other peroxidase- negative granules, ie, specific granules.

scholarly journals Structural and functional heterogeneity among peroxidase-negative granules in human neutrophils: identification of a distinct gelatinase- containing granule subset by combined immunocytochemistry and subcellular fractionation

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3183-3191 ◽  
Author(s):  
L Kjeldsen ◽  
DF Bainton ◽  
H Sengelov ◽  
N Borregaard
Keyword(s):  
Ultrastructural Localization ◽  
Subcellular Fractionation ◽  
Free Form ◽  
Human Neutrophils ◽  
Immunogold Electron Microscopy ◽  
Double Labeling ◽  
Thin Sections ◽  
Dense Granules ◽  
Specific Granules ◽  
Neutrophil Gelatinase

The existence of separate gelatinase granules in human neutrophils has been a matter of debate in recent years. We have demonstrated that the 135-kD form of neutrophil gelatinase is a complex of 92-kD gelatinase and a novel 25-kD protein termed neutrophil gelatinase-associated lipocalin (NGAL) that, in addition to being complexed with part of the gelatinase, is localized in free form in peroxidase-negative specific granules. Because this association was not appreciated in earlier studies, we decided to reassess the ultrastructural localization of gelatinase using specific antibodies without immunoreactivity towards NGAL. Double-labeling immunogold electron microscopy was performed on frozen thin sections of human neutrophils. Twenty-four percent of all peroxidase-negative granules were labeled with antigelatinase antibody, but not with antilactoferrin antibody. These granules are defined as gelatinase granules. Sixteen percent reacted with antilactoferrin antibody but not with antigelatinase antibody. The rest (60%) reacted with both antibodies. All granules labeling for lactoferrin are defined as specific granules. Gelatinase granules were observed as round and oval forms of considerably smaller size than specific granules, and were less electron dense. Isolated granules obtained by subcellular fractionation were also examined by immunoelectron microscopy. This demonstrated that peroxidase-negative granules comprise a continuum from the most dense granules that contain lactoferrin but no gelatinase to the lightest that contain gelatinase but no lactoferrin. Thus, gelatinase granules do exist as a subpopulation of peroxidase-negative granules and may allow for exocytosis of gelatinase during neutrophil diapedesis without substantial mobilization of other peroxidase- negative granules, ie, specific granules.

scholarly journals Celastrol attenuates fMLP-induced superoxide anion generation, myeloperoxidase production, and elastase release by human neutrophils

2021 ◽  
Vol 18 (9) ◽  
pp. 1805-1809
Author(s):  
Nipapan Malisorn ◽  
Ammara Chaikan
Keyword(s):  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Human Neutrophils ◽  
Superoxide Generation ◽  
Xtt Assay ◽  
Dependent Inhibition ◽  
Blood Neutrophils ◽  
Ic50 Values ◽  
Dose Dependent Inhibition ◽  
Anti Inflammatory

Purpose: To investigate the anti-inflammatory effect of celastrol via attenuation of formyl-methionylleucyl-phenylalanine (fMLP)-induced superoxide generation, myeloperoxidase production, and elastase release by peripheral blood neutrophils. Methods: Cytotoxicity of celastrol on human peripheral blood neutrophils was investigated using a 2Htetrazolium hydroxide (XTT) assay. Human neutrophils were stimulated with 100-nM fMLP; the effect of celastrol on superoxide generation was determined via ferricytochrome C reduction, the effect on myeloperoxidase production by tetramethylbenzidine oxidation, and the effect on elastase activity by Boc-Ala-ONp hydrolysis. Results: Treatment of human neutrophils with celastrol showed dose-dependent inhibition of fMLPinduced superoxide generation, myeloperoxidase production, and elastase release with half-maximal inhibitory concentration (IC50) values of 5.9 ± 0.1, 1.9 ± 0.2, and 1.5 ± 0.1 µM, respectively. Conclusion: These results indicate that celastrol possesses anti-inflammatory properties via attenuation of fMLP-induced superoxide generation, myeloperoxidase production, and elastase release by peripheral blood neutrophils.

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